Important previous accomplishments of this project include: #1) In Melchionda et al, J Clin Invest 2005, we demonstrated for the first time that IL-7 therapy can potently augment responses to immunization, thus providing solid evidence that IL-7 should be considered as agent for clinical use in the context of tumor vaccines. #2) Results from the first clinical trial of recombinant human IL-7 (rhIL-7) (Rosenberg et al., J of Immunoth, 2006) provided proof-of-principle that IL-7 dramatically modulates T cell homeostasis in humans and demonstrated that, unlike IL2 (which augments suppressive T cells), IL-7 increases CD4+ T cell numbers without preferentially expanding the suppressive subset. The first major accomplishment of this project during FY2008 was the publication of the results of the second trial of rhIL-7 in humans (Sportes et al, J of Exp Med, 2008; 205;1701) . This trial incorporated extensive biologic studies that provided insight into the basis for the dramatic changes in T cell homeostasis induced by IL-7. We observed that IL-7 causes a broad repertoire of T cells to undergo cell cycling and to resist programmed cell death, resulting in rapid, significant expansion of both blood and tissue T cells. The expansion was tightly regulated by dynamic regulation of the IL-7 receptor, thus preventing dangerous increases in lymphocytes with IL7 therapy. Because IL-7 selectively expands the youngest or most naive T cell populations, T cells in IL-7 treated patients resembled T cells normally present at young ages. Thus, IL-7 has an anti-aging effect on the immune system. This capacity to expand a broad pool of nave cells is unique among cytokines currently under study and is predicted to be very important for immune competence, especially in the context of T cell depletion. Finally, we saw little to no evidence of toxicity with IL-7, providing a solid basis for Phase II trials of this agent in combination with directed immunotherapy and for studies of IL-7 in HIV infection. The second major accomplishment of this project during FY2008 involved the development of an master cell bank of an artificial antigen presenting cells (aAPCs) that effectively expand cytolytic CD8+ T cells and can therefore serve as a critical reagent for developing immune based therapies for cancer. We had previously demonstrated a critical role for 4-1BB mediated costimulation in enhancing immunity toward Ewings Sarcoma (Zhang et al, Cancer Biology and Therapy, 2003) and on this basis investigated approaches to optimize expansion of cytolytic T cells ex vivo that could be used in adoptive immune based therapies for cancer. In collaboration with Drs. Carl June and Bruce Levine at the University of Pennsylvania, we demonstrated that memory CD8+ T cells could be most efficiently expanded by provision of a T cell receptor signal and 4-1BB costimulation using a cell based artificial APC that naturally produced interleukin 15 (IL-15) and expressed IL-15 receptor alpha. Remarkably, provision of CD28 based costimulation at the same time actually diminished the effectiveness of the approach since the preferential expansion of nave populations by CD28 led to a competitive disadvantage for the memory cell populations and eventually resulted in loss of the tumor specific and viral specific T cells which are the focus of most adoptive immune based therapy efforts. This work was published during FY2008 (Zhang et al, J Immunol 2007; 179:4910). Furthermore, it served as the basis for the development of a master cell bank, which is clinical grade and can be used to expand T cells for use in clinical trials. Our plans are to develop such trials during the upcoming year and initiate adoptive cell therapies for childhood tumors as part of our clinical trial project. During FY2008 we also collaborated on a project with Dr. Christoph Raders laboratory to investigate the biology of an anti-NKG2D monoclonal antibody, which can be used to either block or activate NK mediated effects (Kwong et al, J Mol Bio 2008). Using our expertise in NKG2D biology based upon studies of NKG2D expression on CD8+ T cells in lytic activity of Ewings sarcoma cells, we were able to demonstrate that the anti-NKG2D monoclonal developed by the Rader lab was bound specifically to NKG2D and that the Fab fragment could be used to inhibit NKG2D binding to its ligands. Importantly, when crosslinked, this same molecule was also effective at enhancing NKG2D mediated killing, a feature that is potentially exploitable for clinical applications. This molecule will be further studied for its effects in vivo with a particular goal of enhancing antibody dependent cellular cytotoxicity mediated by currently available monoclonal antibodies which target cancer cells, but which are often limited in their capacity to induce antibody dependent cellular cytotoxicity.